Methanogenic potential and methanotrophic activity

The rate of methane production (methanogenesis) was measured using the PMP method (Segers 1998). C-amended solutions (flushed for 5 minutes with N2) with acetate Ca(CH3COO)2 (100 mg C in the incubation flask) were used for the examination of methanogenic potential. All laboratory sediment incubations were performed in 250-mL dark glass flasks, capped with rubber stoppers, using approximately 100 g (wet mass) of sediment (grain size < 1 mm) and 180 mL of amended solution or distilled water. The headspace was maintained at 20 mL. Typically, triplicate live and dead (methanogenesis was inhibited by addition of 1.0 mM chloroform) samples from each depth were stored at 20°C in the dark and the incubation time was 72 hours; however, subsamples from the headspace atmosphere were taken every 24 hours. Gas production was calculated from the difference between final and initial headspace concentration and volume of the flask; results are expressed per volume unit of wet sediment (CH4 mL-1 WW hour-1) or per unit dry weight of sediment per one day (pg CH4 kg-1 DW day-1). Rate of potential methane oxidation (methanotrophy) was measured using modified method of methane oxidation in soil samples from Hanson (1998). Briefly, 50 mL of methane was added by syringe to the closed incubation flask with the sieved sediment and then the pressure was balanced to atmospheric pressure. All laboratory sediment incubations were performed in 250-mL dark glass flasks, capped with rubber stoppers, using approximately 100 g (wet mass) of sediment (grain size < 2 mm). Typically, triplicate live and dead (samples killed by HgCl2 to arrest all biological activity) samples from each depth were stored at 20°C in the dark, and incubation time was 72 hours; however, subsamples from the headspace atmosphere were taken every 24 hours. Potential CH4 oxidation rates at the different concentrations were obtained from the slope of the CH4 decrease with time (r2 > 0.90; methane oxidation was calculated from the difference between final and initial headspace concentration and volume of the flask; results are expressed per volume unit of wet sediment (CH4 mL-1 WW hour-1) or per unit dry weight of sediment per one day (mg CH4 kg-1 DW day-1).